KR101299614B1 - Oxidised phospholipids comprising a fluorophore moiety and use in the determination of the presence of enzymes having antiatherogenetic activity - Google Patents

Abstract

The invention relates to oxidized phospholipids having one of the general formulas (I) or (II) wherein A═O, C, NH, or S; B═O, C, NH, or S; and R2 is selected from the group consisting of —CO—(CH2)n—CH3; —CO—(CH2)n—CHO; and —CO—(CH2)n—COOH, with n=3-7, with the proviso that in general formula (I), R1 is selected from the group consisting of —CH2—(CH2)n—X; and —CO—(CH2)n—X with n=5-11, wherein X is a fluorophore; and in general formula (II), R1 is selected from the group consisting of —CH═CH—(CH2)n—CH3 with n=9-15; —(CH2)n—CH3 with n=11-17; and —CO—(CH2)n—CH3 with n=10—16; and R3 is selected from the group consisting of —CO—(CH2)n—X; and —SO2—(CH2)n—X, with n=0-5, wherein X is a fluorophore.

Description

OXIDISED PHOSPHOLIPIDS COMPRISING A FLUOROPHORE MOIETY AND USE IN THE DETERMINATION OF THE PRESENCE OF ENZYMES HAVING ANTIATHEROGENETIC ACTIVITY

 The present invention relates to oxidized phospholipids represented by either general formula (I) or (II).

Oxidative modification of LDL is considered to be an important pathological factor in atherosclerosis. Studies in several laboratories have shown that the biological effects triggered by mmLDL may be largely due to phospholipid oxidation products (Leitinger, N. et al. (1999) PNAS 96, 12010-12015; Watson, AD et (1995) J. Clin. Invest. 95, 774-782; Leitinger, N. et al. (1997) Adv.Exp. Med. Biol 433, 379-382; Loidl, A. et al. (2003) J. Biol. Chem. 278, 32921-32928). Their concentration increases in the plaque of atherosclerosis (Watson, AD et al. (1997) J. Biol. Chem. 272, 13597-13607; Itabe, H. et al. (1994) J. Biol. Chem 269, 15274-15279) and elevated antibody titers against oxidized phospholipids in lesioned humans and mice (Horkko, S. et al. (1999) J. Clin. Invest. 103, 117-128; Palinski, W.) et al. (1995) Arterioscler. Thromb. Vasc. Biol. 15, 1569-1576; Palinski, W. et al. (1996) J. Clin. Invest. 98, 800-814). Raise interest in relevance

Interestingly increasing interest is two representatives of homogeneous series of oxidized phospholipids: 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2 It is concentrated in-(5-oxovaleroyl) -sn-glycero-3-phosphocholine (POVPC). The importance is further increased by the discovery that they selectively activate processes that may contribute to the development of atherosclerosis in vascular wall cells as well as other chronic inflammatory diseases. Both of these lipids have been demonstrated to be 3-6 fold thickened in atherosclerotic lesions in rabbits, corresponding to about 62 and 116 ng / mg of aortic wetting weight of PGPC and POVPC, respectively (Subbanagounder, G. et al. (2000). Arterioscler.Thromb. Vasc. Biol. 20, 2248-2254).

WO 01/75170 discloses a method for assessing the risk for atherosclerosis, wherein a biological sample comprising HDL is contacted with oxidized phospholipids to check for changes in the amount of oxidized or unoxidized phospholipids. No change in the amount of oxidized phospholipids represents a risk for atherosclerosis.

As mentioned above, oxidized phospholipids participate in the development of atherosclerosis. Oxidized phospholipids are also known to be hydrolyzed by enzymes associated with lipoproteins and anti-atherogenic activity such as phospholipase or PAF acetylhydrolase.

It is an object of the present invention to provide compounds that can be used to collect information about the activity of anti-atherogenic enzymes in a sample. In particular, their interaction with enzymes should result in detectable products that can draw conclusions about the enzyme activity of the sample.

The above object is achieved by oxidized phospholipids represented by either general formula (I) or (II).

Where

A is O, C, NH or S;

B is O, C, NH, or S;

R ₂ is —CO— (CH ₂ ) _n —CH ₃ , _wherein n is 3-7; -CO- (CH ₂ ) _n -CHO; And -CO- (CH ₂ ) _n -COOH,

Provided that in formula (I), R ₁ is —CH ₂ — (CH ₂ ) _n —X, _wherein n is 5-11; And -CO- (CH ₂ ) _n -X, X is a chromophore,

However, in the general formula (II), R ₁ is a -CH = CH- (CH ₂₎ n -CH _{3 n} is 9-15; -(CH ₂ ) _n -CH ₃ , _wherein n is 11-17; And -CO- (CH ₂ ) _n -CH ₃ where n is 10-16;

R ₃ is —CO— (CH ₂ ) _n —X _wherein n is 0-5; And -SO _2- (CH ₂ ) _n -X, X is a chromophore.

In a preferred embodiment, A and / or B are oxygen.

In another preferred embodiment, in general formula (I), X is a chromophore selected from the group consisting of pyrene, perylene and nitrobenzaxadiazole, preferably pyrene.

In another preferred embodiment, in general formula (II), X is pyrene, perylene, borodiazaindacene (BODIP Y ™), cyanine dye 2, cyanine dye 3, cyanine dye 5 and Alexa dye It is selected from the group consisting of.

Examples of preferred oxidized phospholipids according to formula (I) are the following compounds:

1- (10-Pyrendecanoyl) -2-glutaroyl-sn-glycero-3-phosphocholine (PyrGPC)

1- (10-Pyrendecanoyl) -2- (5-oxovaleroyl) -sn-glycero-3-phosphocholine (PyrOVPC)

Preferred examples of oxidized phospholipids according to formula II are the following compounds:

1-palmitoyl-2-glutaroyl-sn-glycero-3-phospho-N- (3- [4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene ] -Propionyl) -ethanolamine (BODIPY-PGPE)

1-palmitoyl-2-glutaroyl-sn-glycero-3-phospho-N- (alexa fluor 647-carbonyl) -ethanolamine (Alexa647-PGPE)

1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-N- (3- [4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene] -Propionyl) -ethanolamine (BODIPY-POPE)

1-palmitoyl-2- (5-oxovaleroyl) -sn-glycero-3-phospho-N- (3- [4,4-difluoro-4-bora-3a, 4a-diaza -s-indacene] -propionyl) -ethanolamine (BODPY-POVPE).

It has been found that the fluorescently labeled oxidized phospholipids according to the invention can be used as substrates for enzymes with anti-atherogenic activity. The claimed compounds are particularly useful for the diagnostic determination of these enzymes in blood samples. Fluorescently labeled cleavage products resulting from the interaction between the oxidized phospholipid and the enzyme under investigation can be easily and sensitively measured by chromatographic analysis such as HPLC, thus providing information on the enzyme activity present in the sample. have.

Another aspect of the invention relates to the use of the oxidized phospholipids according to the invention in determining the presence in a sample of an enzyme having anti-atherogenic activity, preferably phospholipase or PAF-acetylhydrolase.

Another aspect of the invention is the presence of an enzyme with anti-atherogenic activity in the sample, comprising adding an oxidized phospholipid according to the invention to a sample and subjecting the sample to a chromatographic analysis, preferably HPLC. It is about how to determine.

The invention will be explained in more detail by the following examples and accompanying drawings.

1A and B outline the steps for the synthesis of BODIPY-PGPE, BODIPY-POPE and BODPY-POVPE.

2 is a mass spectrum of BODIPY-POVPE, BODIPY-PGPE and BODIPY-POPE.

3 outlines the synthesis steps of PyrGPC and PyrOVPC.

4 is the mass spectrum of PyrGPC and PyrOVPC.

Synthesis of Oxidized Fluorescent Phospholipids

One- Palmy toil -2- Glutaroyl - sn - Glycerol -3- Force -N- (3- [4,4- Difluoro -4-bora-3a, 4a- Diaza -s- Indasen ] - Propionyl ) -Ethanolamine ( BODIPY - PGPE )

1.Synthesis of 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC)

PGPC (FIGS. 1A, 1) are described in Watson et al. (J. Biol. Chem. 272, 13597-13607, 1997) and Subbanagounder et at. (Free Radic. Biol. Med. 28, 1751-1761, 2000). Dry 1-palmitoyl-sn-glycero-3-phosphocholine (62 mg, 125 μmol), dry glutaric anhydride (70 mg, 613 μmol, 5 eq) in 6 mL of anhydrous dichloromethane and DMAP (p- A solution of (N, N'-dimethylamino) pyridine, 75 mg, 614 μmol, 5 eq) was performed overnight at 35 ° C. with magnetic stirring. The reaction was monitored by TLC using CHCl ₃ / Me0H / 25% NH ₃ (65: 35: 5, v / v / v) as the developing system and quenched by addition of 3 mL of MeOH. The resulting mixture was washed once with 1.8 mL of MeOH / H ₂ O (1: 1, v / v). The organic phase was evaporated and then triturated with 4 ml of diethyl ether to remove excess DMAP. The supernatant was removed to give 32 mg of PGPC (2%, R _f = 0.05).

2. Synthesis of 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphoethanolamine (PGPE)

PGPE (FIGS. 1A, 2) is PGPC (90 mg) by PLD (phospholipase D, 29 units) in 4.3 ml emulsion of 0.5 M sodium acetate buffer (pH 7.2) containing 1.4 ml toluene and 0.5 M ethanolamine. , 148 μmol) of phosphatidyl group was obtained by transphosphatidylation (Fig. 1A, step 1). The biphasic system was stirred overnight at 35 ° C. The reaction was monitored by TLC and quenched by addition of 4.3 mL of MeOH. The product was extracted with 43 mL of CHCl ₃ / Me0H (2: 1, v / v). The organic phase was washed twice with 11 ml of MeOH / H ₂ O (1: 1, v / v) to remove excess ethanolamine, then evaporated and preparative TLC was performed. The product was scraped off and eluted from silica gel three times with CHCl ₃ / Me0H (1: 4, v / v). The combined extracts were evaporated to afford the desired product (30.3 mg, 36%, R _f = 0.30).

3. 1-palmitoyl-2-glutaroyl-sn-glycero-3-phospho-N- (3- [4,4-difluoro-4-bora-3a, 4a-diaza-s- Synthesis of indacene] -propionyl) -ethanolamine (BODIPY-PGPE)

BODIPY-SE (4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s in CHCl ₃ / Me0H (2: 1, v / v) stirred with magnetic Indacene-propionic acid succinimidyl ester, 0.96 mg, 2.5 μmol) solution, PGPE (4.4 mg, 7.8 μmol, 3 eq) (FIGS. 1A, 2) and triethylamine pa (10 μL, 72 μmol, 30) eq) (FIG. 1A, step 2) was added. Thereafter, nitrogen was blown into the flask, the light was blocked, and the resulting solution was stirred at room temperature for 1 hour. The progress of the reaction was monitored by TLC. The solvent was removed from the lipids purified by preparative TLC until the volume reached 400 μl under a nitrogen stream. The desired compound was identified under UV light and then TLC was stripped off and eluted from silica gel using CHCl ₃ / Me0H (1: 4, v / v). Solvent was removed from the extracts collected by rotary evaporation to give BODIPY-PGPE (1.77 mg, 85%, R _f = 0.28) (FIG. 1A, 3).

One- Palmy toil -2- Glutaroyl - sn - Glycerol -3- Force -N- ( Alexa647 -Carbonyl) -ethanolamine ( Of Alexa647-PGPE)  synthesis

Alexa647-PGPE corresponds to BODIPY-PGPE (FIGS. 1A, 3) which includes Alexa647 instead of BODIPY in the head group. This was followed by Alexa647-SE (Alexa Fluor 647 carboxylic acid succinimidyl ester, 0.5 mg, 0.4 μmol) and PGPE (0.68 mg, 1.2 μmol, 3 eq in 0.5 mL CHCl ₃ / Me0H (2: 1, v / v). Triethylamine pa (5 [mu] L, 36 [mu] mol, 90 eq) was added and then stirred at room temperature for 90 minutes to obtain. This reaction mixture was thoroughly light blocked. The progress of the reaction was monitored by TLC (RP-18 F254s; Merck, Darmstadt, Germany). The product was purified by preparative TLC using H ₂ O / EtOH / n-propanol (20:57:23, v / v / v) as the development system, scraped off and then CHCl ₃ / Me0H (1: 1, v / eluted twice with v) and once with MeOH. The solvent was removed under nitrogen stream to give Alexa647-PGPE (0.26 μmol, 65%, R _f = 0.80).

One- Palmy toil -2- (5- Oxovaleroyl ) - sn - Glycerol -3- Force -N- (3- [4,4- Difluoro -4-bora-3a, 4a- Diaza -s- Indasen ] - Propionyl ) - Of ethanolamine (BODPY-POVPE)  synthesis

1.Synthesis of 1-palmitoyl-2- (5-dimethoxypentanoyl) -sn-glycero-3-phosphoethanolamine

5,5-dimethoxypentanoic acid methyl ester (220 mg, 1.25 mmol) (FIG. 1B, 4) and sodium hydroxide in 5 mL H ₂ O / MeOH / THF (2: 5: 3, v / v / v) 250 mg, 6.25 mmol, 5 eq) solution was stirred for 90 minutes at room temperature (FIG. 1B, step 1). After cooling to 0 ° C., 6 ml of 1 N and a suitable amount of 0.1 M HCl were added to the reaction mixture, acidified to pH 2.1 and extracted with dichloromethane (3 × 15 ml). The combined organic extracts were washed with water (2 × 10 mL) and dried over Na ₂ SO ₄ . Solvent except 10-15 mL containing the desired product (R _f = 0.35) (FIGS. 1B, 5) was removed under reduced pressure and used directly in the acylation reaction without further purification. 1-palmithyl-sn-glycero-3-phosphocholine (209 mg, 0.42 mmol) (FIG. 1B, 6), DCC (N.N'-dicyclohexylcarbodiimide, 270 mg, 1.3 mmol, 3 eq) and DMAP (160 mg, 1.3 mmol, 3 eq) were added to this 5,5-dimethoxypentanoic acid (FIG. 1B, step 2) solution. The mixture was stirred overnight at room temperature under nitrogen. The reaction was monitored by TLC. 6 mL of MeOH was added, and then the organic solution was washed twice with MeOH / H ₂ O (1: 1, v / v). The solvent was removed in vacuo, benzene / EtOH (3: 2, v / v) was added and water was evaporated. The oily residue was subjected to flash chromatography using CHCl ₃ / Me0H / 25% NH ₃ (65: 35: 5, v / v / v) as solvent on 12 g of silica gel to give 1-palmitoyl- 2- (5-Dimethoxypentanoyl) -sn-glycero-3-phosphocholine (245 mg, 91%, R _f = 0.14) was obtained (FIGS. 1B, 7). As described above for the conversion of PGPC to PGPE (compare FIG. 1A, compared to step 1), phosphatidyl transfer of choline lipids yielded ethanolamine analogs (FIGS. 1B, 8) (yield: 6.2 mg, 66%, R _f = 0.24, using CHCl ₃ / MeOH / 25% NH ₃ , 65: 35: 5, v / v / v as the developing solvent (FIG. 1B, step 3).

2. 1-Palmitoyl-2- (5-oxovaleroyl) -sn-glycero-3-phospho-N- (3- [4,4-difluoro-4-bora-3a, 4a- Synthesis of diaza-s-indacene] -propionyl) -ethanolamine (BODIPY-POVPE)

1-palmitoyl-2- (5-dimethoxypentanoyl) -sn-glycero-3-phosphoethanolamine (3.0 mg, 5.0 μmol) in 1 ml CHCl ₃ / MeOH (2: 1, v / v) Triethylamine pa (10 μl, 72 μmol, 14 eq) was added to the (FIG. 1B, 8) and BODIPY-SE (1.95 mg, 5.0 μmol, 1 eq) solutions (FIG. 1B, step 4). The reaction mixture was stirred at rt for 80 min. 1-palmitoyl-2-dimethoxypentanoyl-sn-glycero-3-phospho-N- (3-BODIPY-propionyl) -ethanol that can be used in the next reaction without further purification by removing the solvent under vacuum An amine was obtained (FIG. 1B, 9). The desired product was isolated by acetal cleavage of the stable precursor (2.0 mg, 2.3 μmol) with 400 μl THF / HCl (1 N) (FIG. 1B, step 5). After only 2 minutes, the reaction mixture was neutralized with NaHCO ₃ and then extracted with 1.2 mL CHCl ₃ / MeOH (2: 1, v / v). The organic phase was washed twice with 300 μl of water, dried over Na ₂ SO ₄ and evaporated under reduced pressure to give 1.65 mg of BODIPY-POVPE (87%, R _f = 0.22, CHCl ₃ / MeOH / H ₂ 0 with development system). , 15: 5: 0.1, using v / v / v) (FIG. 1B, 10).

One- Palmy toil -2- Oleo oil - sn - Glycerol -3- Force -N- (3- [4,4- Difluoro -4-bora-3a, 4a- Diaza -s- Indasen ] - Propionyl ) - Of ethanolamine (BODIPY-POPE)  synthesis

BODIPY-SE (1.00 mg, 2.57 μmol), triethylamine pa (10 μl, 72 μmol, 28 eq) and POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 5.53 mg, 7.70 μmol, 3 eq) was dissolved in 1 mL CHCl ₃ / MeOH (2: 1, v / v). The mixture was stirred at room temperature for 60 minutes and then the solvent was removed under nitrogen stream. The white oily residue was dissolved in 500 μl CHCl ₃ / MeOH (2: 1, v / v). The product was purified by preparative TLC. Fluorescent bands containing the product were scraped off the plate and eluted three times with CHCl ₃ / MeOH (1: 4, v / v). The combined extracts were evaporated to afford the desired product (1.31 mg, 51%, R _f = 0.59).

1- (10- Pyrendecanoyl )-2- Glutaroyl - sn - Glycerol -3- Of phosphocholine (PyrGPC)  synthesis

1,2-bis (10) stirred with magnetic in a mixture of 3 ml of 0.1 M Tris-HCl buffer (pH 8) containing 0.1 M CaCl ₂ and 3 ml of diethylether (peroxide-deficient) To the pyrendecanoyl) -sn-glycero-3-phosphocholine (45 mg, 47 μmol) (FIGS. 3, 11) emulsion, 50 units of PLA ₂ (Naja naja venom) were added (FIG. 3, step 1). ). The reaction mixture was stirred overnight at 35-40 ° C. After diethyl ether was removed, the product was extracted from the aqueous solution using CHCl ₃ / Me0H (2: 1, v / v) (3 × 5 mL). The combined organic fractions are evaporated and the remaining water is removed under high vacuum to give a mixture of 1- (10-pyrendecanoyl) -sn-glycero-3-phosphocholine (Figures 3, 12) and free pyrendecanoic acid 50 Mg was obtained. The latter was removed by chewing with diethyl ether. Removal of solvent in vacuo gave 25 mg of pure lysophospholipid (88%, R _f = 0.12, CHCl ₃ / MeOH / AcOH / H ₂ O, 50: 30: 10: 5, v / v / v / v as a deployment system). Glutar anhydride (12 mg, 105 μmol, 11 eq) and anhydrous DMAP (4 mg, 33 μmol, 3 eq) were added to a solution of lysophospholipid (6.0 mg, 9.8 μmol) in 3 mL of anhydrous dichloromethane ( 3, step 2a). The reaction was stirred overnight at 35-40 ° C. Flash chromatography of the crude product with CHCl ₃ / Me0H (/ H ₂ O (65: 25: 4, v / v / v) on 10 g of silica gel gave PyrGPC (2.2 mg, 31%, R _f = 0.18, CHCl ₃ / MeOH / AcOH / H ₂ O, 50: 30: 10: 5, using v / v / v / v as the development system) were obtained (FIG. 3, 14).

1- (10- Pyrendecanoyl ) -2- (5- Oxovaleroyl ) - sn - Glycerol -3-phosphocholine ( PyrOVPC ) Synthesis of

1- (10-pyrendecanoyl) -sn-glycero-3-phosphocholine (10 mg, 16 μmol) (FIGS. 3, 12) was added to DCC (200 mg, 1.0 mmol, 59 eq) and DMAP (200 mg). , Acylated with 5,5-dimethoxypentanoic acid (acid "5") in 10 ml of dichloromethane containing 1.6 mmol, 100 eq). The reaction mixture was stirred overnight at room temperature (FIG. 3, step 2b). The progress of the reaction was monitored by TLC until the reaction was stopped by adding 6 ml of MeOH. The resulting solution was washed twice with MeOH / H ₂ O (1: 1, v / v) and the solvent was removed under reduced pressure. The crude product was purified by preparative TLC, scraped off and eluted five times with 4 ml of CHCl ₃ / MeOH (1: 4, v / v). The remaining silica gel was removed by washing the combined organic fractions (concentrated to 5 mL) with 1 mL of MeOH / H ₂ O (1: 1, v / v). Evaporation of the solvent gave 1- (10-pyrendecanoyl) -2- (5-dimethoxypentanoyl) -sn-glycero-3-phosphocholine (49%, R _f = 0.17) (FIG. 3, 15). Deprotection of intermediate 15 (5.6 mg, 7.4 μmol) was carried out by acetal cleavage with THF / HCl (1 N), then neutralized with NaHCO ₃ , and then the product was CHCl ₃ / MeOH (2: 1, v / v). The organic phase was washed twice, dried over Na ₂ SO ₄ and evaporated under reduced pressure to give 1.1 mg of PyrOVPC (21%, R _f = 0.05) (FIG. 3, step 3).

Mass spectrometry

Matrix assisted laser desorption / ionisation time-of-flight (MALDI-TOF) mass spectra were obtained on a Micromass TofSpec2E equipped with a nitrogen laser (λ = 337 nm, operating at 5 Hz) and a time-lag focusing unit. Was performed. Irradiation was slightly higher than the threshold laser power to generate ions. Spectra were recorded in reflectron mode with an accelerating voltage of 20 kV and calibrated externally with a titration mixture of poly (ethylene glycol) (PEG). The sample is typically a substrate (2,5-dihydroxybenzoic acid, c = 10 mg / ml, CH ₃ Cl / 0.1% trifluoroacetic acid (TFA), 70:30, v / v), analyte ( c = 0.01-1 mg / ml, CHCl ₃ / MeOH, 2: 1, v / v) and NaTFA (c = 1 mg / ml, CH ₃ CN / H ₂ O, 70:30, v / v) It was prepared by mixing in a 1: 1: 0.5 (v / v / v) ratio. 0.5 μl fractions from this mixture were placed on sample plates (stainless steel) and dried in air. Spectra of 50-100 shots were averaged to improve the signal to noise ratio. All m / z values discussed in this experiment are the strongest peaks in all isotope distributions. Mass spectra of BODIPY-POVPE, BODIPY-PGPE and BODIPY-POPE are shown in FIG. 2. Mass spectra of PyrGPC and PyrOVPC are shown in FIG. 4.

Claims (15)

Oxidized phospholipid represented by one of the general formulas (I) or (II):

Where A is O, C, NH or S; B is O, C, NH, or S; R ₂ is —CO— (CH ₂ ) _n —CH ₃ , _wherein n is 3-7; -CO- (CH ₂ ) _n -CHO; And -CO- (CH ₂ ) _n -COOH, Provided that in formula (I), R ₁ is —CH ₂ — (CH ₂ ) _n —X, _wherein n is 5-11; And -CO- (CH ₂ ) _n -X, X is a chromophore, However, in the general formula (II), R ₁ is a -CH = CH- (CH ₂₎ n -CH _{3 n} is 9-15; -(CH ₂ ) _n -CH ₃ , _wherein n is 11-17; And -CO- (CH ₂ ) _n -CH ₃ where n is 10-16; R ₃ is —CO— (CH ₂ ) _n -X, wherein n is 0-5; And -SO _2- (CH ₂ ) _n -X, X is a chromophore. The oxidized phospholipid represented by the general formula (I) according to claim 1, wherein A and / or B are oxygen. The oxidized phospholipid represented by the general formula (I) according to claim 1 or 2, wherein X is a fluorophore selected from the group consisting of pyrene, perylene and nitrobenzisadiazole. The compound of formula (I) according to claim 1 or 2, wherein the compound is 1- (10-pyrendecanoyl) -2-glutaroyl-sn-glycero-3-phosphocholine (PyrGPC). Oxidized Phospholipids Displayed. 3. General according to claim 1, characterized in that 1- (10-pyrendecanoyl) -2- (5-oxovaleroyl) -sn-glycero-3-phosphocholine (PyrOVPC). 4. Oxidized phospholipid represented by formula (I). An oxidized phospholipid represented by the general formula (I) according to claim 1 or 2, which is used to determine the presence of an enzyme having antiatherogenetic activity in a sample. A method for preparing an enzyme having anti-atherogenic activity in a sample, the method comprising adding to the sample an oxidized phospholipid represented by the general formula (I) according to claim 1 or performing a chromatographic analysis of the sample. How to determine the existence. The oxidized phospholipid represented by the general formula (II) according to claim 1, wherein A and / or B are oxygen. The compound of claim 1 or 8, wherein X is composed of pyrene, perylene, borodiazaindacene (BODIP Y ™), cyanine dye 2, cyanine dye 3, cyanine dye 5, and Alexa dye. An oxidized phospholipid represented by the general formula (II), which is a chromophore selected from the group. 9-Palmitoyl-2-glutaroyl-sn-glycero-3-phospho-N- (3- [4,4-difluoro-4-bora-3a according to claim 1 or 8. , 4a-diaza-s-indacene] -propionyl) -ethanolamine (BODIPY-PGPE), oxidized phospholipid represented by the general formula (II). The compound according to claim 1 or 8, which is 1-palmitoyl-2-glutaroyl-sn-glycero-3-phospho-N- (alexa fluoro 647-carbonyl) -ethanolamine (Alexa647-PGPE). Oxidized phospholipid represented by the general formula (II). The method of claim 1 or 8, wherein 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-N- (3- [4,4-difluoro-4-bora-3a, Oxidized phospholipid represented by the general formula (II), characterized in that it is 4a-diaza-s-indacene] -propionyl) -ethanolamine (BODIPY-POPE). The compound of claim 1 or 8, wherein 1-palmitoyl-2- (5-oxovaleroyl) -sn-glycero-3-phospho-N- (3- [4,4-difluoro- Oxidized phospholipid represented by the general formula (II), characterized as 4-bora-3a, 4a-diaza-s-indacene] -propionyl) -ethanolamine (BODPY-POVPE). An oxidized phospholipid represented by the general formula (II) according to claim 1 or 8, which is used to determine the presence of an enzyme having anti-atherogenic activity in a sample. A method for preparing an enzyme having anti-atherogenic activity in a sample, comprising adding to the sample an oxidized phospholipid represented by formula (II) according to claim 1 or 8. How to determine the existence.

Images